Étude de l'implication des cellules souches de la zone sous-ventriculaire dans la récupération post-hypoxie néonatale

Perinatal hypoxia leads to degeneration and delayed maturation of oligodendrocytes and cortical glutamatergic neurons. My PhD project consists in assessing the contribution of neural stem cells (NSCs) of the dorsal subventricular zone (dSVZ, i.e. the largest germinal zone of the postnatal brain) to the spontaneous regenerative attempt observed following such injury as well as its amenability to pharmacological manipulation.The results I have obtained highlight a dynamic and lineage-specific response of NSCs of the dSVZ to hypoxia that results in de novo oligodendrogenesis and cortical neurogenesis. Newborn cortical neurons express appropriate cortical layer markers, supporting their appropriate specification. A pharmacogenomics analysis allowed us to identify small molecules boosting specificly dSVZ NSCs. Pharmacological activation of Wnt/ß-catenin signalling by intranasal GSK3ß inhibitor administration during the recovery period following hypoxia indeed potentiates dorsal SVZ participation to post-hypoxia repair. Gsk3b inhibitor CHIR99021 seems to promote survival of cortical neurons from the dSVZ produced in response to hypoxia. More interestingly, CHIR99021 promotes oligodendrocyte maturation and long term integration in the cortex as well as a long term increased activity of dSVZ NSCs.Altogether, my results highlighted a dynamic and lineage-specific response of dorsal NSCs cells to hypoxia and identify the early postnatal dorsal SVZ as a malleable source of stem cells for cortical repair following trauma that occur early in life. CHIR99021 (a Gsk3b inhibitor) intranasal administration promotes this cortical cellular repair with a long term activation of dSVZ NSCs which increased their production of oligodendrocytes migrating to the cortex and a short term improvement of their maturation, and might allow the integration of cortical neurons they produceL'hypoxie périnatale entraîne une dégénérescence et un délai de maturation des oligodendrocytes et des neurones corticaux du cortex cerebral. Mon projet de thèse a d'abord consisté à étudier la contribution des cellules souche neurales de la zone sous-ventriculaire dorsale (dSVZ) à la tentative de régénération spontanée observée après la lésion. Dans un second temps, j'ai étudié la capacité de ces cellules souches à être manipulée en utilisant une approche pharmacologique.Mes résultats mettent en évidence une réponse spontanée et dynamique de la dSVZ qui produit des neurones et des oligodendrocytes corticaux en réponse à l'hypoxie. L'administration par voie intranasale d'un inhibiteur de Gsk3b, qui active la voie Wnt/b-caténine, petite molécule identifiée à l'aide d'une étude bio-informatique comme « dorsalisante », juste après la période d'hypoxie, potentialise cette réponse spontanée. En effet, mes résultats montrent que certains neurones corticaux issus de la dSVZ survivent avec le traitement alors qu'aucun ne semblent persister après 1 mois suivant l'hypoxie. De plus, le traitement accélère la maturation des oligodendrocytes corticaux et augmentent leur production et intégration à long terme. Enfin, le traitement a un effet à long terme sur les cellules souches de la dSVZ en augmentant la proportion de ces cellules qui sont actives. Pour conclure, la dSVZ participe à la récupération corticale spontanée qui suit l'hypoxie périnatale et cette réponse peut être potentialisée par l'administration d'une petite molécule identifiée par notre analyse bio-informatique, un inhibiteur de GSK3

Nkd1 is sufficient to antagonize ectopic Wnt8a.

<p>Overexpression of Wnt8a (25pg) results in an eyeless phenotype that can be rescued by co-injection of <i>nkd1</i> (A, B) which is quantified in (C). Numbers above each column represent n values. Overexpression of high Wnt8a (200pg) results in ectopic <i>gsc</i> expression along the ventral-lateral domain at 50% epiboly (E). Co-injection of high <i>wnt8a</i> with <i>nkd1</i> mRNAs dramatically reduces the ectopic <i>gsc</i> expression, but leaves the putative endogenous <i>gsc</i> domain intact (F).</p

Nkd1 does not influence normal development and patterning of the early embryo.

<p>Injection of <i>nkd1</i> mRNA or morpholino (MO) does not have an obvious affect during early somitogenesis (A–F) or at 1 dpf (G–I). The number and width of somites in injected individuals is indistinguishable from uninjected embryos. At 1 dpf, injection of <i>nkd1</i> MO results in neural necrosis, which ranges from moderate (H) to more severe (I).</p

Nkd1 does not rescue the <i>hdl</i>/<i>tcf7l1a</i> mutant.

<p>Homozygous deletion of <i>tcf7l1a</i> results in an eyeless phenotype due to activated canonical Wnt signaling (D). Overexpression of Nkd1 in embryos from a homozygous hdl-/- parental cross (B, E) does not rescue the eyeless phenotype (E) and does not affect development of the early embryo (B), although at 1 dpf, <i>nkd1</i> injected embryos typically have a kinked axis. Injection of <i>nkd1</i> MO into embryos from a <i>hdl</i> +/- X <i>hdl</i> -/- parental cross has no affect on early (C) or 1 dpf (F) development (Uninj <i>hdl</i> +/- n= 14, <i>hdl</i> -/-n= 16; Nkd1 MO injected <i>hdl</i> +/- n= 15, <i>hdl</i> -/-n=22). Experiments in embryos from homozygous hdl-/- parental crosses had similar results (not shown).</p

The <i>knypek</i> (glypican 6) mutant is not sensitive to Nkd1.

<p>Knockdown of Nkd1 in <i>kny</i> -/- (C, D) or in <i>kny</i> +/+; +/- (G, H) does not have any affect on the <i>kny</i> mutant phenotype during somitogenesis (A–H) or at 1 dpf (I; n=19 kny-/-). Note the high levels of neural necrosis in <i>nkd1</i> MO injected embryos at 1 dpf (I). Consistent with the lack of sensitivity, overexpression of Nkd1 has no obvious effects during early somitogenesis (J–M) or at 1 dpf (N; n=31 <i>kny</i> -/-). There is no change in the ratio of wild-type: mutant phenotypes.</p

Nkd1 is insufficient to antagonize constitutively active β-catenin.

<p>Overexpression of ∆N-β-catenin results in a spectrum of dorsal-ventral (D–V) phenotypes ranging from a severe phenotype (A), which shows dramatic reduction in both dorsal and ventral structures to a moderate phenotype (B), which have reduced dorsal and ventral structures. The addition of Nkd1 does not ameliorate the effect of ∆N-β-catenin. The distribution of phenotypes in <i>∆N-β-catenin</i> and <i>nkd1</i> injections is quantified in (D), with numbers above each column representing n values. Consistent with the 1 dpf phenotype, ∆N-β-catenin overexpression results in expansion of <i>gsc</i> expression (E, F), which is not reduced by the addition of Nkd1 (G) (uninj n=40; <i>∆N-</i>β-<i>catenin</i> n=32; <i>∆N-</i>β-<i>catenin+nkd1</i> n=34).</p

The <i>silberblick</i> (<i>Wnt11</i>) mutant is sensitive to Nkd1.

<p>The <i>slb</i> mutant undergoes normal convergence and extension (A), which is not affected by knockdown of Nkd1 with morpholinos (B). Overexpression of Nkd1 in slb-/- reduces <i>gsc</i> (D) and <i>chd</i> (F) expression (arrows), relative to controls (C, E) at 50% epiboly (Nkd1 injected: 47% of embryos with reduced <i>chd</i> expression (n=81); 50% of embryos with reduced <i>gsc</i> expression (n=46)). In contrast, knockdown of Nkd1 results in a slight expansion of <i>gsc</i> expression at 30% epiboly (G: ave <i>gsc</i> width=0.35 mm; n=13, H: ave <i>gsc</i> width=0.38 mm; n=22). All embryos are homozygous <i>slb</i>, derived from homozygous <i>slb</i> parents.</p

Trilobite (<i>vangl2</i>) mutants are highly sensitive to canonical signaling.

<p>Injection of a low dose (5pg) of <i>wnt8a</i> into embryos from a tri+/- X tri+/- parental cross results in extremely dorsalized embryos and significant lethality (A–D, I). Different classes of phenotypes are shown in (A–D) with an uninjected wild-type sibling shown at the top in (A), and an uninjected tri mutant shown at the top in (B) for comparison. Addition of Nkd1 is capable of fully suppressing the <i>wnt8a</i> overexpression lethality and dorsalization (I). n values are for all genotypes. In contrast to the extreme phenotypes seen at 1 dpf, there is no effect of Wnt8a on the early organizer (E–H) shown by expression of <i>gsc</i> (E, F) and <i>bozozok</i> (<i>boz</i>) (G, H) an early and direct transcription target of maternal Wnt signaling. (J–M) Injection of <i>vangl2</i> MO does not alter the expression of <i>gsc</i> (K), nor does the low level of <i>wnt8a</i> (M). However, co-injection of <i>vangl2</i> MO and <i>wnt8a</i> results in ectopic <i>gsc</i> expression about 2-5% of the time (red arrowheads, n= 21).</p

<i>Trilobite</i> (<i>vangl2</i>) mutants are sensitive to Nkd1.

<p>Knockdown of Nkd1 with morpholinos (D, E) or overexpression of Nkd1 (I, J) does not have an obvious effect during early somitogenesis. However, at 1 dpf, knockdown or overexpression of Nkd1 in <i>tri</i> mutants results in an increase in cyclopia (K, L, O). Before the onset of gastrulation, at 50% epiboly, embryos generated from a tri+/- X tri+/- parental cross are sensitive to Nkd1 overexpression, demonstrated by a reduction or absence of <i>gsc</i> expression (M; n=32, N; 56% of embryos with reduced expression, n=25). (O) The cyclopic index was calculated using criteria established in Marlow et al., 1998 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074666#B43" target="_blank">43</a>]. n values reflect the number of tri-/- embryos. Error bars represent standard error.</p